PROJECT SUMMARY The lack of knowledge about the interactions among cardiac contractile regulatory proteins represents an important problem because it not only limits our understanding of how such interactions affect cardiac thin- filament activation, but it also precludes an understanding of mechanisms underlying many forms of heart disease. Cardiac muscle contraction depends on coordinated interactions among contractile regulatory pro- teins, which include cardiac troponin C (cTnC), troponin T (cTnT), troponin I (cTnI), and tropomyosin (Tm). Coordinated interactions among these proteins play key roles during contraction via Ca2+-, strong cros- sbridge (XB)-, and sarcomere length (SL)-mediated activation of thin filaments. However, very little is known about how cTnT influences the actions of Tm, cTnI, and cTnC to modulate Ca2+-, SL-, and XB- acti- vation of cardiac thin filaments. Our long-term goal, therefore, is to determine how structural differences in contractile regulatory proteins determine Ca2+-, SL-, and strong XB-mediated activation of cardiac muscle contraction, and how they are altered in heart disease. The overall objective of this proposal is to determine how cTnT interacts with Tm, cTnI, and cTnC to modulate cardiac thin-filament activation by Ca2+, SL, and strong XB. Our hypothesis is that the structural features of the tail (cT1) and the head (cT2) domains of cTnT are the key determinants of functional features of cardiac thin-filament activation. To test our hypo- thesis, we will measure force/ATPase, rate of tension redevelopment and myofiber dynamic stiffness in re- constituted cardiac muscle fibers. Further, complementary studies such as Ca2+ binding kinetics measure- ments and quantitative mathematical modeling will be performed. Specific Aim 1 will determine how cT1 modulates the dynamics of strong XB recruitment during cardiac thin-filament activation. This aim will be accomplished by determining: the specific region of cT1 that affects thin-filament activation; how changes in the overlapping ends of contiguous Tm impact cT1 effects on cardiac thin-filament activation; and how cT1 effects on thin-filament activation are modified by myosin isoforms. Specific Aim 2 will determine how cTnT modulates the cooperative feedback of strong XB on conformational changes in cTnC during cardiac thin- filament activation. In this aim, we will determine how key regions of cT2 modulate Ca2+- and XB-induced changes in cTnC structure. Specific Aim 3 will determine how interactions between cTnT and cTnI mediate the feedback effect of strong XB on cardiac thin-filament activation. This aim will be accomplished by de- termining: how cT2-cTnI interactions modulate cardiac thin-filament activation and how differences in myo- sin isoforms alter cT2-cTnI effect on thin-filament activation. The expected outcome from our comprehen- sive and multidisciplinary approach will have a positive impact because it will significantly advance our un- derstanding of the molecular mechanisms underlying cardiac thin-filament activation. Our study will lay a strong foundation for developing novel pharmacological strategies aimed at improving cardiac function in diseased hearts.